ATP-dependent AAA+ proteases remove toxic proteins from cells and regulate many other important cellular processes that are required to promote health and prevent disease. Because protein degradation is irreversible, it must be carefully regulated. The architectures of AAA+ proteases and the principles of degradation control are similar in all organisms. AAA+ proteases assemble into multi- subunit structures with an internal proteolytic chamber, accessible through narrow channels that exclude natively folded proteins. This mechanism protects most proteins from unintended degradation and requires specific substrates to be recognized, unfolded, and then translocated into the degradation chamber. In the AAA+ ClpXP protease, for example, a ring hexamer of ClpX unfolds specific target proteins and translocates them into ClpP for degradation. ClpXP is one of the best-characterized AAA+ proteases and is a paradigm for other ATP-dependent proteases and non-proteolytic AAA+ enzymes. These ATP-fueled molecular machines perform mechanical work in the cell, including protein and nucleic-acid remodeling (e.g., helicases), transport of cargo along microtubules, secretion and vesicle recycling, cell-cycle control, viral budding, cytokinesis, activation of apoptosis and the innate immune response, chromosome translocation, viral DNA packaging, peroxisome biogenesis, transcriptional activation, and clamp and helicase loading onto DNA. In humans, mutations in many AAA+ proteins are linked to disease. For example, mutations in the m-AAA mitochondrial protease result in hereditary spastic paraplegia. ClpXP can also promote bacterial pathogenesis and is an antibiotic target. Substantial progress has been made in understanding the general biochemical and structural features of ClpXP and other AAA+ enzymes but important and fundamental questions concerning the molecular mechanisms of these intracellular machines remain unanswered. For instance, in no case, do we understand the ATPase cycle of a AAA+ enzyme and how it is linked to the cycle of conformational changes that power machine function or whether ATP hydrolysis occurs in a sequential or probabilistic fashion. The experiments described in this proposal will answer these questions for ClpXP and provide a conceptual framework and a set of novel tools applicable to studies of the entire superfamily of AAA+ machines. Specifically, we will use biochemical, protein-engineering, and single-molecule approaches to determine how ATP binding, ATP hydrolysis, and the coordination of these reactions among the six subunits of a ClpX ring drive the conformational changes that allow this enzyme to mechanically unfold and translocate protein substrates and to cooperate with ClpP.